This invention relates generally to methods for the desorption and analysis of surface adsorped species. More specifically, this invention relates to desorption by selective bond cleavage using resonant energy sources such as electromagnetic radiation and particle beams.
The advent of new technologies based on chemical reactions on solid supports has generated an ever increasing need for accurate, efficient and selective chemical analysis of atoms and molecules adsorbed onto the solid. Research and development in this area is primarily concentrated in combinatorial chemistry and ordered monolayer films (e.g. Langmuir-Blodgett and self-assembled monolayers). Depending on the technological application, these adsorbed species can be inorganic or organic compounds as well as a large array of biomolecules including proteins, peptides, oligonucleotides, DNA fragments and pharmaceutical compounds. Selective chemical analysis of surface adsorbed species often presents special difficulties since these molecules are bound to the surface and, hence, any process which attempts to remove or desorb the surface molecules has a high probability of dissociating or fragmenting the sought after species. It is often difficult to identify the original molecular species with data produced from fragmented and degraded molecules.
One method that has been used for analyzing surface adsorbed biomolecules is laser induced desorbtion followed by mass spectrometric analysis of the desorbed species. Variations on this method are described in U.S. Pat. Nos. 5,719,060, 5,118,937, 4,920,264, 5,062,935 and 4,988,879, all of which are incorporated herein by reference. While the methods disclosed in these patents have proved useful in analysis of surface adsorbed biomolecules, none of the methods include the selective cleavage of molecules from a solid surface using resonant excitation processes. As described below, such a selective, resonant method has important advantages over the methods described in these patents.
U.S. Pat. No. 5,719,060 to Hutchens, which is hereby incorporated by reference, discloses the adsorption of analyte molecules onto a probe surface followed by their subsequent laser induced desorption using ultra-violet (UV) radiation and detection using mass spectrometry. Hutchens does not disclose selective cleavage of specific adsorbate/substrate bonds using resonant energy excitation. One important drawback of using non-resonant, UV radiation is that the analyte molecules can directly absorb this radiation which could lead to fragmentation of the desorbed species. It is often critically important to avoid fragmentation of the desorbed species since this makes subsequent analysis much more difficult. A second limitation of the techniques of Hutchens and others involved in matrix assisted laser desorption/ionization (described below) is that the analyte species may resist ionization because of its inherent properties such as its ionization potential, proton affinity, hydride affinity or electron affinity.
U.S. Pat. Nos. 5,118,937, 5,062,935 and 4,920,264, each of which are hereby incorporated by reference, disclose methods in which the analyte molecules are incorporated into a matrix that absorbs laser light. In these methods, the matrix absorbs the laser light which heats the matrix and leads to thermal desorption and ionization of the analyte molecules. This ionization occurs primarily by charge transfer (electron or positive or negative ion), cationization or electron ejection processes. These ions of the analyte molecules may then be analyzed using standard mass spectrometric methods. The main drawback of these matrix-assisted methods is that the analyte molecules must form a mixture with the matrix such that the analyte can desorb with the matrix. In addition, these methods are not useful for a monolayer or few monolayers of analyte bound to a solid support.
U.S. Pat. No. 4,988,879 to Zare, et al. discloses an apparatus and method for laser desorption and volatilization of molecules and is here by incorporated by reference. In this method, analyte molecules are physisorbed onto a solid support that can absorb laser light. When irradiated with the desorbing laser, the support absorbs the laser light which heats the support and leads to thermal desorption of the analyte molecules. This method is a thermal desorption process which does not attempt to selectively desorb the analyte molecules. In addition, this method could lead to extensive fragmentation of thermally sensitive biomolecules which could significantly impede their identification with the method.
In sum, the previously disclosed methods require either a matrix or support that can absorb laser light to induce thermal desorbtion and are all limited to non-selective or non-resonant laser excitation. The present invention is a new method for analysis of surface adsorbed molecules that overcomes the drawbacks of these previous methods. Specifically, the present invention allows the desorption of intact molecules as opposed to fragments. The method should achieve a very high yield of desorbed, intact moleculesxe2x80x94one or more orders of magnitude higher than any other known technique. Using the present method the chemical noise in the analysis of the desorbed, intact molecules may be orders of magnitude lower than with existing techniques, permitting, in principle, orders of magnitude higher detection limits for molecules bound to surfaces.
The present invention provides a method for the selective, specific chemical analysis of molecules bound or bonded to solid surfaces. The general method includes the steps of (1) selecting an electromagnetic wavelength that is approximately resonant with an energy transition between two or more energy states of the chemical bonds at the surface of an absorbate/substrate system and (2) irradiating the surface of this absorbate/substrate system with the electromagnetic radiation to effect selective dissocation or bond breakage of specific chemical bonds at or near the surface of the absorbate/substrate system. The method can include the additional step of detecting the absorbate by mass spectrometry.
The selective cleavage of a bond in the adsorbate/substrate system may be accomplished by irradiating the system with electromagnetic radiation or by bombarding the system with a particle beam. In one embodiment, the system may be both irradiated with electromagnetic radiation and bombarded with a particle beam.
In the embodiment using electromagnetic radiation, the wavelength of the radiation is selected to be approximately resonant with a transition between two energy levels in the adsorbate/substrate system. These energy levels may be separated by an electronic, vibrational, or rotational transition or some combination of these transitions. Irradiation of the adsorbate/substrate system with electromagnetic radiation at the selected wavelength will lead to cleavage of a bond in the adsorbate/substrate system and desorption of the adsorbate. It is preferred to use a laser to irradiate the adsorbate/substrate system and it is more preferred to use a pulsed laser. It is most preferred to use a laser in the infra-red region of the spectrum.
The bond cleaved in adsorbate/substrate system by the resonant excitation can be due to physisorption, ionic or covalent(chemisorption) interactions between the analyte molecule and elements or molecules in the substrate. Bond cleavage can be one or more of the bonds of the absorbate to the substate or a bond removed from the adsorbate/substrate bond. Alternatively, the cleaved bond can be a bond within the substrate that is at a position removed from the adsorbate/substrate bond.
Generally, any substrate capable of forming physisorbed, ionic or covalent bonds with the adsorbate may be used.
Preferred substrates include glass, metal, semiconductor, and carbonaceous substrates. A preferred substrate is glass, in which case the adsorbate may be bonded to the substrate via one or more silicon atoms and/or one or more oxygen atoms. In a preferred embodiment, the adsorbate contains a silicon atom bonded to one or more silicon oxide moieties on the substrate surface. This may be represented as Sxe2x80x94Sixe2x80x94Oxe2x80x94Sixe2x80x94R where S represents the remainder of the substrate and R represents the remainder of the adsorbate. In this embodiment, a preferred resonant transition to be excited is the asymmetric stretch of the Sixe2x80x94Oxe2x80x94Sixe2x80x94 group. Excitation of this transition may lead to cleavage of either the substrate/adsorbate, xe2x80x94Oxe2x80x94Sixe2x80x94R bond or bonds within the substrate. In this embodiment of the invention, it is preferred that wavelength selected is between about 8.85 micrometers and about 10.00 micrometers. It is also preferred that the laser is a pulsed laser with a pulse width of less than about one millisecond.
In an alternative embodiment the resonant excitation may be accomplished by bombarding the adsorbate/substrate system with a particle beam having an energy approximately equal to the transition between the energy levels in the adsorbate/substrate system. Suitable particles include electrons, positrons, positive elemental ions, negative elemental ions, neutral atoms, positive molecular ions, negative molecular ions, positive cluster ions, negative cluster ions, and neutral molecules.